Blood glucose concentration is a important marker for diabetes diagnosis. In addition, quantification of glucose concentration is used in monitoring the process of fermentative production using microorganisms. Conventionally, glucose quantification is performed by an enzymatic method using glucose oxidase (GOD) or glucose-6-phosphate dehydrogenase (G6PDH). However, the GOD method requires addition of catalase or peroxidase into the assay system to quantify hydrogen peroxide levels generated by oxidative reaction of glucose. G6PDH has been used for glucose quantification based on spectroscopy. This method involves the addition of coenzyme NAD(P) into the assay system.
Recently the application of PQQGDH, an enzyme which uses pyrroloquinoline quinine as a coenzyme is attracting attention, in place of the enzyme used in the existing glucose quantification method. PQQGDH is a glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, and catalyzes the reaction of oxidizing glucose to produce gluconolactone.
Two types of PQQGDHs are known: membrane-bound and water-soluble. Membrane-bound PQQGDH is a single-peptide protein with an approximate molecular weight of 87 kDa, and is found in a wide variety of gram-negative bacteria. See, for example, J. Bacteriol. (1990) 172, 6308-6315, A. M. Cleton-Jansen et al. On the other hand, water-soluble PQQGDH has been found in some strains of Acinetobacter calcoaceticus (Biosci. Biotech. Biochem. (1995), 59 (8), 1548-1555), and its structural gene has been cloned and its amino acid sequence determined (Mol. Gen. Genet. (1989), 217:430-436). Water-soluble PQQGDH derived from A. calcoaceticus is a homodimer enzyme with an approximate molecular weight of 50 kDa. It shows little homology in primary structure with other PQQ enzymes.
Recently, the results of X-ray structural analysis of water-soluble PQQGDH were published and its conformation and active center were revealed (A. Oubrie, et al. (1999) J. Mol. Bio., 289, 319-333, A. Oubrie, et al. (1999) The EMBO Journal, 18 (19), 5187-5194, and A. Oubrie, et al. (1999), PNAS 96 (21), 11787-11791). These reports demonstrate that water-soluble PQQGDH is a β-propeller protein consisting of six W-motifs.
PQQGDH is expected to have potential in glucose assays, for example, as a recognition device of a glucose sensor, because it has highly oxidative activity towards glucose and does not require oxygen as an electron acceptor as it is complexed with a coenzyme. However, the low selectivity of PQQGDH for glucose was an obstacle to this use.
The object of this invention is to provide a modified water-soluble PQQGDH with a high selectivity for glucose.